Mutant light-inducible ion channel of chrimson

ABSTRACT

The invention relates to mutant light-inducible ion channels having improved properties as compared to the parent channel, nucleic acid constructs encoding same, expression vectors carrying the nucleic acid construct, cells comprising the nucleic acid construct or expression vector, and their respective uses, as well as non-human animals comprising the mutant light-inducible ion channel, the nucleic acid construct or the expression vector as disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International Application PCT/EP2017/063458, filed Jun. 2, 2017, which designates the U.S. and was published by the International Bureau in English on Dec. 7, 2017, and which claims the benefit of European Patent Application No. 16172984.3, filed Jun. 3, 2016; both of which are hereby incorporated herein in their entirety by reference.

The invention relates to mutant light-inducible ion channel having improved properties as compared to the parent light-inducible ion channel, nucleic acid constructs encoding same, expression vectors carrying the nucleic acid construct, cells comprising said nucleic acid construct or expression vector, and their respective uses, as further defined in the claims.

BACKGROUND OF THE INVENTION

The light-gated, inwardly rectifying cation channels, channelrhodopsin-1 (ChR1) and channelrhodopsin 2 (ChR2) has become a preferred tool for the targeted light-activation of neurons both in vitro and vivo¹⁻⁵. Although wild-type (WT) ChR2 can be employed for light-induced depolarization, there is an ongoing search for ChR2 mutants with faster kinetics and increased light-sensitivity for potential future clinical applications (WO 03/084994 and⁶⁻⁸).

Since the first description in 2002 and 2003 a set of different variants of ChR2 including a red-light absorbing channelrhodopsin are described. For different purposes ChR's were modified with respect to the kinetics, ion selectivity as well as light absorption. Examples are the red light absorbing channelrhodopsin from Chlamydomonas Chrimson (WO 2013/071231 and⁹; accession number KF992060), the Chrimson variant ChrimsonR (K176R), and CsChrimson, a chimeric polypeptide comprising the amino acid sequence from Chlamydomonas Chrimson, and an amino acid sequence derived from a Chloromonas channelrhodopsin CsChR, See also SEQ ID NOs: 1, 2, and 5 of US 2016/0039902, and accession number KJ995863. Red light activated channelrhodopsins are beneficial, because the penetration depths of red light into animal tissues is deeper than the penetration depths of lower wavelength light (^(3,8)). Moreover the use of red light activated channelrhodopsins reduces the risk of phototoxicity.

The kinetics are a major issue, because the light sensitivity is regulated via the open time of the channel. This is due to the invariance of other channel parameters like single channel conductance, open probability, quantum efficiency. In other words, channels with a long open time reach the maximal activity at low light intensity, whereas short living channels need more light to reach saturation with respect to light saturation. Although ‘fast’ channels need more light for the activation, high speed is indispensable for many applications in neurobiology because of the high frequency firing rate of different neuronal cells. This is valid e.g. for ganglion cells in the auditory system for interneurons in the brain, which reach firing rates up to 1000 Hz. Accordingly, there is still a need for mutant light-inducible ion channels combining robust expression and faster response kinetics.

SUMMARY OF THE INVENTION

The inventors performed a systematic study on Chrimson by modifications in helix 6 of the seven transmembrane helix motif. Helix 6 movement during light-activation is a common feature in microbial-type rhodopsins (^(15,17)). Therefore helix 6 was modified in order to change the closing time of the channel. It could be demonstrated that mutation of positions 261, 267, and 268 in helix 6 in Chrimson accelerates the closing time (off-kinetics) of the channel and that the combination of the mutations leads to a further acceleration of the off-kinetics.

The present disclosure describes a general way to modify Chrimson with respect to speed by specific point mutations in helix 6. The use of these new variants will provide a light stimulation of neurons up to their limits of 800 to 1000 Hz.

An experimental verification for the increased speed was tested in NG108-15 cells (neuroblastoma cells), in HEK293 cells, and hippocampal cells from the mouse brain.

Accordingly, disclosed is a mutant light-inducible ion channel, wherein the mutant light-inducible ion channel comprises an amino acid sequence which has at least 90% similarity/homology and/or at least 72% identity to the full length sequence of SEQ ID NO: 1 (Chrimson), and wherein the mutant light-inducible ion channel only differs from its parent light-inducible ion channel by a substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1,

which substitution(s) accelerate(s) the off-kinetics of the mutant channel as compared to the parent channel, when compared by patch-clamp measurements in the whole cell configuration at a clamp potential of −60 mV, a bath solution of 140 mM NaCl, 2 mM CaCl₂, 2 MgCl₂, 10 mM HEPES, pH 7.4, and a pipette solution of 110 mM NaCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.4.

Further provided is a nucleic acid construct, comprising a nucleotide sequence coding for the light-inducible ion channel as disclosed herein.

Also provided is an expression vector, comprising a nucleotide sequence coding for the light-inducible ion channel or the nucleic acid construct as disclosed herein. Moreover, a cell is provided, comprising the nucleic acid construct or the expression vector as disclosed herein.

In still another aspect, the invention provides the use of the light-inducible ion-channel disclosed herein in a high-throughput screening, and/or for stimulating neurons. The use of the mutant light-inducible ion channel in medicine is also contemplated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present disclosure pertains to mutant light-inducible ion channel, wherein the mutant light-inducible ion channel comprises an amino acid sequence which has at least 90% similarity/homology and/or at least 72% identity to the full length sequence of SEQ ID NO: 1 (Chrimson), and wherein the mutant light-inducible ion channel only differs from its parent light-inducible ion channel by a substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1,

which substitution(s) accelerate(s) the off-kinetics of the mutant channel as compared to the parent channel, when compared by patch-clamp measurements in the whole cell configuration at a clamp potential of −60 mV, a bath solution of 140 mM NaCl, 2 mM CaCl₂, 2 MgCl₂, 10 mM HEPES, pH 7.4, and a pipette solution of 110 mM NaCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.4. The off-kinetics of the mutant and the parent channel can be measured in rat hippocampal cells (see Example 3 below), or in NG108-15 neuroblastoma cells (see Example 1 below), each heterologously expressing the mutant or parent channel. Preferably, the off-kinetics of the mutant channel are measured in NG108-15 cells. Successful protein expression can be proven, for example, by EGFP- or YFP-mediated fluorescence. Generally, photocurrents are measured in response 3 ms light pulses with an intensity of 23 mW/mm² and a wavelength of 594 nm. The T_(off) value is determined by a fit of the current after cessation of illumination to a monoexponential function, as further described in the examples below.

In a preferred embodiment, the mutant light-inducible ion channel comprises a substitution at position Y261. In a particularly preferred embodiment, the substitution is Y261F.

Alternatively, or in addition to the substitution at position Y261, the mutant light-inducible ion channel comprises a substitution at position Y268. More preferably, the substitution is Y268F.

In still another preferred embodiment, the mutant light-inducible ion channel comprises a substitution at position S267. In a particularly preferred embodiment, the substitution is S267M. Even more preferably, said substitution is combined with the substitution at position Y261, at position Y268, or both at positions Y261 and Y268.

Hence, in a preferred embodiment, the mutant light-inducible ion channel comprises a substitution at position Y261 and at position S267, preferably wherein the substitution at position Y261 is Y261F, and preferably wherein the substitution at position S267 is S267M. In another preferred embodiment, the mutant light-inducible ion channel comprises a substitution at position Y268 and at position S267, preferably wherein the substitution at position Y268 is Y268F, and preferably wherein the substitution at position S267 is S267M. In still another preferred embodiment the mutant light-inducible ion channel comprises a substitution at position Y261, at position Y268, and at position S267, preferably wherein the substitution at position Y261 is Y261F, preferably wherein the substitution at position Y268 is Y268F, and preferably wherein the substitution at position S267 is S267M.

The parent light-inducible ion channel may be any Chrimson-like channel, as long as it falls within the required percentage sequence identity and/or sequence homology/similarity. In one preferred embodiment, the parent light-inducible ion channel already comprises an Arg at the position corresponding to position 176 of SEQ ID NO: 1. Said variant is already known as ChrimsonR (K176R).

Preferably, the mutant light-inducible ion channel has at least 91%, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% similarity/homology to the full length of SEQ ID NO: 1 (Chrimson).

In addition, or alternatively, the mutant light-inducible ion channel has at least 74%, preferably at least 75%, more preferably at least 76%, more preferably at least 78%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity to the full length of SEQ ID NO: 1 (Chrimson).

Examples of light-inducible ion channels, which have at least 70% similarity/homology or at least 70% identity to the full length of SEQ ID NO: 1 are the ChrimsonR variant K176R, or the chimera CsChrimson (SEQ ID NO: 2, accession number KJ995863), and any other ortholog or allelic variant thereof. Chrimson and CsChrimson share 74% identity and 76.4% homology/similarity over the full length of Chrimson (SEQ ID NO: 1).

Preferably, the mutant light-inducible ion channel is a red light absorbing channelrhodopsin.

Also contemplated are swap mutants of a light-inducible ion channel, in which helix 6 has been replaced by the helix-6 motif of SEQ ID NO: 3, e.g. ChR-2 (SEQ ID NO: 5), VChR1 (SEQ ID NO: 6), or ReaChR (SEQ ID NO: 7) in which helix 6 is replaced by helix 6 of Chrimson including the Y268F, Y261F, and S267M substitutions, respectively (cf. SEQ ID NO: 8, 9, and 10).

Such a swap mutant has typically at least 56% homology/similarity to the full length of SEQ ID NO: 1 (Chrimson), preferably at least 58%, more preferably at least 60%, more preferably at least 62%, and even more preferably at least 64% homology/similarity to the full length of SEQ ID NO: 1 (Chrimson).

In addition, or alternatively, such a swap mutant has typically at least 42%, preferably at least 44%, more preferably at least 46%, more preferably at least 48%, more preferably at least 50%, more preferably at least 52%, more preferably at least 54%, more preferably at least 56% identity to the full length of SEQ ID NO: 1 (Chrimson).

Generally, an amino acid sequence has “at least x % identity” with another amino acid sequence, e.g. SEQ ID NO: 1 above, when the sequence identity between those to aligned sequences is at least x % over the full length of said other amino acid sequence, e.g. SEQ ID NO: 1. Similarly, an amino acid sequence has “at least x % similarity/homology” with another amino acid sequence, e.g. SEQ ID NO: 1 above, when the sequence similarity/homology between those to aligned sequences is at least x % over the full length of said other amino acid sequence, e.g. SEQ ID NO: 1.

Such alignments can be performed using for example publicly available computer homology programs such as the “EMBOSS” program provided at the EMBL homepage (available on the World Wide Web at ebi.ac.uk/Tools/psa/emboss_needle/) using the default settings provided therein. Further methods of calculating sequence identity or sequence similarity/homology percentages of sets of amino acid sequences are known in the art.

However, in a particularly preferred embodiment, the mutant light-inducible ion channel comprises, more preferably consists of the amino acid sequence of SEQ ID NO: 1 (Chrimson), except for said substitution(s) at position Y261, Y268, and 5267, and optionally the Arg at the position corresponding to position 176 of SEQ ID NO: 1, as further disclosed above.

The light inducible ion channel of the present disclosure is a membrane protein with at least 5 transmembrane helices, which is capable of binding a light-sensitive polyene. Transmembrane proteins with 6 or 7 transmembrane helices are preferable. Transmembrane proteins with more than 7 helices, for example 8, 9 or 10 transmembrane helices, are however also encompassed. Furthermore, the invention covers transmembrane proteins which in addition to the transmembrane part include C- and/or N-terminal sequences, where the C-terminal sequences can extend into the inside of the lumen enclosed by the membrane, for example the cytoplasm of a cell or the inside of a liposome, or can also be arranged on the membrane outer surface. The same applies for the optionally present N-terminal sequences, which can likewise be arranged both within the lumen and also on the outer surface of the membrane. The length of the C- and/or N-terminal sequences is in principle subject to no restriction; however, light-inducible ion channels with C-terminal sequences not embedded in the membrane, with 1 to 1000 amino acids, preferably 1 to 500, especially preferably 5 to 50 amino acids, are preferred. Independently of the length of the C-terminal sequences, the N-terminal located sequences not embedded in the membrane preferably comprise 1 to 500 amino acids, especially preferably 5 to 50 amino acids. The concept of the transmembrane helix is well known to the skilled person. These are generally α-helical protein structures, which as a rule comprise 20 to 25 amino acids. However, depending on the nature of the membrane, which can be a natural membrane, for example a cell or plasma membrane, or also a synthetic membrane, the transmembrane segments can also be shorter or longer. For example, transmembrane segments in artificial membranes can comprise up to 30 amino acids, but on the other hand also only a few amino acids, for example 12 to 16.

In addition, the light-inducible ion channel comprises further (semi-)conservative substitutions as compared to SEQ ID NO: 1. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc. Typical semi-conservative and conservative substitutions are:

Conservative Amino acid substitution Semi-conservative substitution A G; S; T N; V; C C A; V; L M; I; F; G D E; N; Q A; S; T; K; R; H E D; Q; N A; S; T; K; R; H F W; Y; L; M; H I; V; A G A S; N; T; D; E; N; Q H Y; F; K; R L; M; A I V; L; M; A F; Y; W; G K R; H D; E; N; Q; S; T; A L M; I; V; A F; Y; W; H; C M L; I; V; A F; Y; W; C; N Q D; E; S; T; A; G; K; R P V; I L; A; M; W; Y; S; T; C; F Q N D; E; A; S; T; L; M; K; R R K; H N; Q; S; T; D; E; A S A; T; G; N D; E; R; K T A; S; G; N; V D; E; R; K; I V A; L; I M; T; C; N W F; Y; H L; M; I; V; C Y F; W; H L; M; I; V; C

Furthermore, the skilled person will appreciate that glycines at sterically demanding positions should not be substituted and that proline should not be introduced into parts of the protein which have an alpha-helical or a beta-sheet structure.

Preferably, the mutant channel comprises the helix 6-motif of SEQ ID NO: 3:

Cys-Arg-Met-Val-Val-Lys-Leu-Met-Ala-Tyr-Ala-Xaa₁₂-Phe-Ala-Ser-Trp-Gly-Xaa₁₈-Xaa₁₉-Pro-Ile-Leu-Trp-Ala-Val,

wherein Xaa₁₂ is Phe or Tyr, preferably wherein Xaa₁₂ is Phe;

wherein Xaa₁₈ is Met or Ser, preferably wherein Xaa₁₈ is Met; and

wherein Xaa₁₉ is Tyr or Phe, preferably wherein Xaa₁₉ is Phe.

It is further preferred that the light-inducible ion channel comprises the consensus motif L(I,A,C)DxxxKxxW(F,Y) (SEQ ID NO: 4). Amino acids given in brackets can in each case replace the preceding amino acid. This consensus sequence is the motif surrounding the retinal-binding amino acid lysine.

In general, the retinal or retinal derivative necessary for the functioning of the light-inducible ion channel is produced by the cell to be transfected with said ion channel. Depending on its conformation, the retinal may be all-trans retinal, 11-cis-retinal, 13-cis-retinal, or 9-cis-retinal. However, it is also contemplated that the mutant light-inducible ion channel of the invention may be incorporated into vesicles, liposomes or other artificial cell membranes. Accordingly, also disclosed is a channelrhodopsin, comprising the light-inducible ion channel of the present disclosure, and a retinal or retinal derivative. Preferably, the retinal derivative is selected from the group consisting of 3,4-dehydroretinal, 13-ethylretinal, 9-dm-retinal, 3-hydroxyretinal, 4-hydroxyretinal, naphthylretinal; 3,7,11-trimethyl-dodeca-2,4,6,8, 10-pentaenal; 3,7-dimethyl-deca-2,4,6,8-tetraenal; 3,7-dimethyl-octa-2,4,6-trienal; and 6-7 rotation-blocked retinals, 8-9 rotation-blocked retinals, and 10-11 rotation-blocked retinals.

The present disclosure also describes a nucleic acid construct, comprising a nucleotide sequence coding for the mutant light-inducible ion channel as disclosed herein above.

To ensure optimal expression, the coding DNA can also be suitably modified, for example by adding suitable regulatory sequences and/or targeting sequences and/or by matching of the coding DNA sequence to the preferred codon usage of the chosen host. The targeting sequence may encode a C-terminal extension targeting the light-inducible ion channel to a particular site or compartment within the cell, such as to the synapse or to a post-synaptic site, to the axon-hillock, or the endoplasmic reticulum. The nucleic acid may be combined with further elements, e.g., a promoter and a transcription start and stop signal and a translation start and stop signal and a polyadenylation signal in order to provide for expression of the sequence of the mutant light-inducible ion channel of the present disclosure. The promoter can be inducible or constitutive, general or cell specific promoter. An example of a cell-specific promoter is the mGlu6-promotor specific for bipolar cells. Selection of promoters, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

Also disclosed is an expression vector, comprising a nucleotide sequence coding for the mutant light-inducible ion channel or the nucleic acid construct as disclosed herein. In a preferred embodiment, the vector is suitable for gene therapy, in particular wherein the vector is suitable for virus-mediated gene transfer. The term “suitable for virus-mediated gene transfer” means herein that said vector can be packed in a virus and thus be delivered to the site or the cells of interest. Examples of viruses suitable for gene therapy are retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, pox viruses, alphaviruses, rabies virus, semliki forest virus and herpes viruses. These viruses differ in how well they transfer genes to the cells they recognize and are able to infect, and whether they alter the cell's DNA permanently or temporarily. However, gene therapy also encompasses non-viral methods, such as application of naked DNA, lipoplexes and polyplexes, and dendrimers.

As described above, the resulting nucleic acid sequence may be introduced into cells e.g. using a virus as a carrier or by transfection including e.g. by chemical transfectants (such as Lipofectamine, Fugene, etc.), electroporation, calcium phosphate co-precipitation and direct diffusion of DNA. A method for transfecting a cell is detailed in the examples and may be adapted to the respective recipient cell. Transfection with DNA yields stable cells or cell lines, if the transfected DNA is integrated into the genome, or unstable (transient) cells or cell lines, wherein the transfected DNA exists in an extrachromosomal form. Furthermore, stable cell lines can be obtained by using episomal replicating plasmids, which means that the inheritance of the extrachromosomal plasmid is controlled by control elements that are integrated into the cell genome. In general, the selection of a suitable vector or plasmid depends on the intended host cell.

Therefore, the present disclosure also pertains to a cell comprising the nucleic acid construct or the expression vector as disclosed herein.

The incorporation of the mutant light-inducible ion channel into the membrane of cells which do not express the corresponding channels in nature can for example be simply effected in that, using known procedures of recombinant DNA technology, the DNA coding for this ion channel is firstly incorporated into a suitable expression vector, e.g. a plasmid, a cosmid or a virus, the target cells are then transformed with this, and the protein is expressed in this host. Next, the cells are treated in a suitable manner, e.g. with retinal, in order to enable the linkage of a Schiffs base between protein and retinal.

The expression of the light-inducible ion channel of the present disclosure can be advantageously effected in certain mammalian cell systems. Thus, in a preferred embodiment, the cell is a mammalian cell. The expression is effected either with episomal vectors as transient expression, preferably in neuroblastoma cells (e.g., NG108-15-Cells), melanoma cells (e.g., the BLM cell line), COS cells (generated by infection of “African green monkey kidney CV1” cells) or HEK cells (“human embryonic kidney cells”, e.g. HEK293 cells), or BHK-cells (“baby hamster kidney cells”), or in the form of stable expression (by integration into the genome) in CHO cells (“Chinese hamster ovary cells”), myeloma cells or MDCK cells (“Madine-Darby canine kidney cells”) or in Sf9 insect cells infected with baculoviruses. Accordingly, in a more preferred embodiment the mammalian cell is a COS cell; a BHK cell; a HEK293 cell; a CHO cell; a myeloma cell; or a MDCK cell.

In a preferred embodiment, the mammalian cell is an electrically excitable cell. It is further preferred that the cell is a hippocampal cell, a photoreceptor cell; a retinal rod cell; a retinal cone cell; a retinal ganglion cell; a bipolar neuron; a ganglion cell; a pseudounipolar neuron; a multipolar neuron; a pyramidal neuron, a Purkinje cell; or a granule cell.

A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signalling, wherein chemical signalling occurs via synapses, specialized connections with other cells. A number of specialized types of neurons exist such as sensory neurons responding to touch, sound, light and numerous other stimuli affecting cells of the sensory organs, motor neurons receiving signals from the brain and spinal cord and causing muscle contractions and affecting glands, and interneurons connecting neurons to other neurons within the same region of the brain or spinal cord. Generally, a neuron possesses a soma, dendrites, and an axon. Dendrites are filaments that arise from the cell body, often extending for hundreds of microns and branching multiple times. An axon is a special cellular filament that arises from the cell body at a site called the axon hillock. The cell body of a neuron frequently gives rise to multiple dendrites, but never to more than one axon, although the axon may branch hundreds of times before it terminates. At the majority of synapses, signals are sent from the axon of one neuron to a dendrite of another. There are, however, many exceptions to these rules: neurons that lack dendrites, neurons that have no axon, synapses that connect an axon to another axon or a dendrite to another dendrite, etc. Most neurons can further be anatomically characterized as unipolar or pseudounipolar (dendrite and axon emerge from same process), bipolar (axon and single dendrite on opposite ends of the soma), multipolar (having more than two dendrites and may be further classified as (i) Golgi I neurons with long-projecting axonal processes, such as pyramidal cells, Purkinje cells, and anterior horn cells, and (ii) Golgi II: neurons whose axonal process projects locally, e.g., granule cells.

A photoreceptor cell, is a specialized neuron found in the retina that is capable of phototransduction. The two classic photoreceptors are rods and cones, each contributing information used by the visual system. A retinal ganglion cell is a type of neuron located near the inner surface of the retina of the eye. These cells have dendrites and long axons projecting to the protectum (midbrain), the suprachiasmatic nucleus in the hypothalamus, and the lateral geniculate (thalamus). A small percentage contribute little or nothing to vision, but are themselves photosensitive. Their axons form the retinohypothalamic tract and contribute to circadian rhythms and pupillary light reflex, the resizing of the pupil. They receive visual information from photoreceptors via two intermediate neuron types: bipolar cells and amacrine cells. Amacrine cells are interneurons in the retina, and responsible for 70% of input to retinal ganglion cells. Bipolar cells, which are responsible for the other 30% of input to retinal ganglia, are regulated by amacrine cells. As a part of the retina, the bipolar cell exists between photoreceptors (rod cells and cone cells) and ganglion cells. They act, directly or indirectly, to transmit signals from the photoreceptors to the ganglion cells.

The cell may be isolated (and genetically modified), maintained and cultured at an appropriate temperature and gas mixture (typically, 37° C., 5% CO2), optionally in a cell incubator as known to the skilled person and as exemplified for certain cell lines or cell types in the examples. Culture conditions may vary for each cell type, and variation of conditions for a particular cell type can result in different phenotypes. Aside from temperature and gas mixture, the most commonly varied factor in cell culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factor and the presence of other nutrient components among others. Growth media are either commercially available, or can be prepared according to compositions, which are obtainable from the American Tissue Culture Collection (ATCC). Growth factors used for supplement media are often derived from animal blood such as calf serum. Additionally, antibiotics may be added to the growth media. Amongst the common manipulations carried out on culture cells are media changes and passaging cells. The present disclosure further pertains to a use of a mutant light-inducible ion channel, or a cell according to the present disclosure in a high-throughput screening. A high-throughput screening (HTS), is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. HTS allows a researcher to effectively conduct millions of biochemical, genetic or pharmacological tests in a short period of time, often through a combination of modern robotics, data processing and control software, liquid handling devices, and sensitive detectors. By this process, one may rapidly identify active agents which modulate a particular biomolecular pathway; particularly a substance modifying an ion channel, such as the light-inducible ion channel according to the invention, a Ca⁺⁺-inducible potassium channel, or a BK channel. For example, one might co-express the Ca⁺⁺-inducible potassium channel and the light-inducible ion channel in a host cell. Upon stimulation by light, the light-inducible channel will open and the intracellular Ca⁺⁺ concentration will increase, thereby activating the potassium channel. Thus, one will receive a change in the membrane potential, which may be monitored by potential-sensitive dyes such as RH 421 (N-(4-Sulfobutyl)-4-(4-(4-(dipentylamino)phenyl)butadienyl)pyridinium, inner salt). Such a HTS may thus comprise the following steps: (i) contacting a cell expressing a Ca⁺⁺-inducible (potassium) channel and the light-inducible ion channel according to the invention with a candidate agent directed against the Ca⁺⁺-inducible channel, (ii) applying a light stimulus in order to induce the light-inducible channel, (iii) determining the alteration of the membrane potential (mixed signal), and (iv) comparing the signal determined in step (iii) with the signal determined in a cell only expressing the light-inducible ion channel according to the invention subjected to step (ii) (single signal). A reduction in the change of the membrane potential would be indicative of a promising modulator of the Ca⁺⁺-inducible (potassium) channel. Such an approach is supposed to yield a signal-to-noise ratio of approximately 5:1, which is quite improved compared to direct measurements conducted on a cell only expressing the Ca⁺⁺-inducible channel. Due to the improved signal-to-noise ratio, said method, in particular by using the light-inducible ion channel, may be particularly suitable for HTS.

In essence, HTS uses an approach to collect a large amount of experimental data on the effect of a multitude of substances on a particular target in a relatively short time. A screen, in this context, is the larger experiment, with a single goal (usually testing a scientific hypothesis), to which all this data may subsequently be applied. For HTS cells according to the invention may be seed in a tissue plate, such as a multi well plate, e.g. a 96-well plate. Then the cell in the plate is contacted with the test substance for a time sufficient to interact with the targeted ion channel. The test substance may be different from well to well across the plate. After incubation time has passed, measurements are taken across all the plate's wells, either manually or by a machine and optionally compared to measurements of a cell which has not been contacted with the test substance. Manual measurements may be necessary when the researcher is using patch-clamp, looking for effects not yet implemented in automated routines. Otherwise, a specialized automated analysis machine can run a number of experiments on the wells (such as analysing light of a particular frequency or a high-throughput patch-clamp measurement). In this case, the machine outputs the result of each experiment e.g. as a grid of numeric values, with each number mapping to the value obtained from a single well. Depending upon the results of this first assay, the researcher can perform follow up assays within the same screen by using substances similar to those identified as active (i.e. modifying an intracellular cyclic nucleotide level) into new assay plates, and then re-running the experiment to collect further data, optimize the structure of the chemical agent to improve the effect of the agent on the cell. Automation is an important element in HTS's usefulness. A specialized robot is often responsible for much of the process over the lifetime of a single assay plate, from creation through final analysis. An HTS robot can usually prepare and analyze many plates simultaneously, further speeding the data-collection process. Examples for apparatuses suitable for HTS in accordance with the present invention comprise a Fluorometric Imaging Plate Reader (FLIPR™; Molecular Devices), FLEXstation™ (Molecular Devices), Voltage Ion Probe Reader (VIPR, Aurora Biosciences), Attofluor® Ratio Vision® (ATTO).

Thus, the presently disclosed mutant light-inducible ion channel is particularly useful as a research tool, such as in a non-therapeutic use for light-stimulation of electrically excitable cells, in particular neuron cells. Further guidance, e.g., with regard to Hippocampal neuron culture, and electrophysiological recordings from hippocampal neurons, as well as electrophysiological recordings on HEK293 cells, can be found in WO 2012/032103.

Finally, there are a number of diseases in which, e.g., the natural visual cells no longer function, but all nerve connections are capable of continuing to operate. Today, attempts are being made in various research centres to implant thin films with artificial ceramic photocells on the retina. These photocells are intended to depolarise the secondary, still intact cells of the retinal and thereby to trigger a nerve impulse (bionic eyes). The deliberate expression of mutant light-controlled ion channels according to the present disclosure in these ganglion cells, amacrine cells or bipolar cells would be a very much more elegant solution and enable greater three-dimensional visual resolution. Therefore, the present disclosure also contemplates the light-inducible ion channel according to the present disclosure for use in medicine.

The proof of principle is already demonstrated in the examples below, and can easily be adapted to the respective purpose. In view of these data, it is contemplated that the presently disclosed light-inducible ion channels can be used for restoring auditory activity in deaf subjects, or recovery of vision in blind subjects. More specifically, as demonstrated in the examples below, the mutant light-inducible ion channels of the present disclosure provide sufficient temporal fidelity. Their red-shifted spectrum is advantageous for deeper light penetration and less scattering in the tissue as well as less risk of phototoxity. Therefore, it is anticipated that the presently disclosed mutant light-inducible ion channels will become a valuable tool for optogenetic hearing restoration and auditory research. In still another embodiment, it is contemplated that the presently disclosed light-inducible ion channels can be used in afferent feedback for improving sensory-motor prosthetic of limbs, such as an arm or a leg. Afferent feedback refers to nerve signals sent from the peripheral nerves of the body to the brain or spinal cord. It is further contemplated that the presently disclosed light-inducible ion channels can be used in the treatment of pain, in particular phantom limb pain, or chronic pain.

Further described are non-human animals which functionally express the light-inducible ion channel according to the present disclosure, e.g. in an electrically excitable cell such as a neuron, in particular in spiral ganglion neurons, as also described for the cell of the present disclosure. Likewise, also contemplated are non-human animals, which comprise a cell according to the present disclosure.

The non-human animal may be any animal other than a human. In a preferred embodiment, the non-human animal is a vertebrate, preferably a mammal, more preferably a rodent, such as a mouse or a rat, or a primate.

In particular, some model organisms are preferred, such as Caenorhabditis elegans, Arbacia punctulata, Ciona intestinalis, Drosophila, usually the species Drosophila melanogaster, Euprymna scolopes, Hydra, Loligo pealei, Pristionchus paciflcus, Strongylocentrotus purpuratus, Symsagittifera roscoffensis, and Tribolium castaneum. Among vertebrates, these are several rodent species such as guinea pig (Cavia porcellus), hamster, mouse (Mus musculus), and rat (Rattus norvegicus), as well as other species such as chicken (Gallus gallus domesticus), cat (Felis cattus), dog (Canis lupus familiaris), Lamprey, Japanese ricefish (Oryzias latipes), Rhesus macaque, Sigmodon hispidus, zebra finch (Taeniopygia guttata), pufferfish (Takifugu rubripres), african clawed frog (Xenopus laevis), and zebrafish (Danio rerio). Also preferred are non-human primates, i.e. all species of animals under the order Primates that are not a member of the genus Homo, for example rhesus macaque, chimpanzee, baboon, marmoset, and green monkey. However, these examples are not intended to limit the scope of the invention.

It is noted that those animals are excluded, which are not likely to yield in substantial medical benefit to man or animal and which are therefore not subject to patentability under the respective patent law or jurisdiction. Moreover, the skilled person will take appropriate measures, as e.g. laid down in international guidelines of animal welfare, to ensure that the substantial medical benefit to man or animal will outweigh any animal suffering.

In the following, the present invention is illustrated by figures and examples which are not intended to limit the scope of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1: Off-kinetics of Chrimson and Chrimson Y261F. Shown are typical photo currents of Chrimson-YFP and Chrimson-YFP Y261F immediately after cessation of illumination. The currents were normalized for comparison.

FIG. 2: Photocurrents of Chrimson and Chrimson mutants. Shown are typical photocurrents which were measured in response to 3 ms light-pulses with a wavelength of 594 nm and a saturating light intensity of 23 mW/mm⁻². NG108-15 cells which were heterologously expressing Chrimson-YFP (

), Chrimson-YFP K176R (

) Chrimson-YFP S267M (

) Chrimson-YFP Y268F (

), Chrimson-YFP Y261F (

), Chrimson-YFP 5267M/Y268F (

) Chrimson-YFP Y261F/S267M (

) Chrimson-YFP K176R/S267M/Y268F (

) Chrimson-YFP Y261F/S267M/Y268F (

) Chrimson-YFP K176R/Y261F/S267M (

) or Chrimson-YFP K176R/Y261F/S267M/Y268F (

) were investigated by patch-clamp measurements in the whole cell configuration as described in the Examples below. The currents were normalized for comparison.

FIG. 3: Actionspectra of Chrimson and Chrimson mutants. Shown are formed peak currents in response to ns light-pulses of indicated wavelength. NG108-15 cells which were heterologously expressing Chrimson-YFP (

, n=6), Chrimson-YFP S267M/Y268F (

, n=3), Chrimson-YFP S267M (

, n=3), Chrimson-YFP Y261F (

, n=4), Chrimson-YFP Y268F (

, n=5), Chrimson-YFP K176R/Y261F/S267M (

, n=4) or Chrimson-YFP Y261F/S267M (

, n=4) were investigated by patch-clamp measurements in the whole cell configuration as further described in the examples below.

FIG. 4: Spiking traces at different light-pulse frequencies. Rat hippocampal neurons heterologously expressing A) Chrimson-YFP, B) Chrimson-YFP K176R/Y261F/S267M and C) Chrimson-YFP Y261F/S267M were investigated by patch-clamp experiments in the whole cell configuration, as further described in the examples below.

FIG. 5: Postnatal transduction of SGNs with AAV2/6-hSyn-Chrimson-YFP Y261F/S267M is highly efficient and mostly specific to the injected ear. A: Confocal images shown as maximum intensity projections (Z-step: 1 μm) of YFP and calretinin immunofluorescence in three different regions of the spiral ganglion. Scale bar: 50 μm. B: Quantification of YFP expression in SGNs calculated as a ratio to calretinin⁺ SGNs (left panel) and SGNs viability shown as cells/10⁴ μm² (right panel). Symbols mark results from individual animals. Grey and black bars represent average values from the left (injected) and from the right (control) cochleae. No statistically significant differences were found within the same group (injected or control) in both quantifications, neither between groups for the same cochlear region in the cell viability quantification (t-test, p>0.05).

FIG. 6: Chrimson-YFP Y261F/S267M enables optogenetic coding of information at physiological rates of hundreds per second. A: oABR traces (0-2.5 ms) from 4 mice injected with AAV6-Chrimson-YFP Y261F/S267M using a 594 nm light stimulus at maximum intensity (11 mW, 1 ms at 10 Hz). B: oABR traces (0-8 ms) from an exemplary mouse injected with AAV6-Chrimson-YFP Y261F/S267M recorded at increasing laser intensities (1 ms at 10 Hz). Symbols mark P1 and N1 (landmarks for response quantification) on oABR traces (A, B), and help identifying data points from the same mouse through A-F. C: Increase in P1-N1 amplitude (normalized for maximum amplitude) with increasing laser intensity (1 ms at 10 Hz). Group average is shown in black. D: Increase in P1-N1 amplitude (normalized for maximum amplitude) with increasing light pulse duration (5.5 mW at 10 Hz for the animal marked with filled circles, 11 mW at 20 Hz for the rest). Group average is shown in black. E: Decrease in P1 latency with increasing laser intensity (1 ms at 10 Hz). Group average is shown in black. F: Decrease in P1-N1 amplitude (normalized for maximum amplitude) with increasing stimulus rate (5.5 mW, 1 ms for the animals marked with diamonds and empty circles; 6.6 mW, 1 ms for the rest). Group average is shown in black. Shaded area in C-F indicate +/−standard deviation.

DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 Chrimson; accession number KF992060; helix 6 highliqhted in bold) MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSY GLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAI ALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCL RYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLK WLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYP ILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTK EIGGEEVEVEEFVEEEDEDTV SEQ ID NO: 2 (CsChrimson; accession number KJ995863; helix 6 highlighted in bold) MSRLVAASWLLALLLCGITSTTTASSAPAASSTDGTAAAAVSHYAMNGFDELAKG AVVPEDHFVCGPADKCYCSAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFS AWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLS CPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIY GGYMYFQAAKCYVEANHSVPKGHCRMWKLMAYAYFASWGSYPILWAVGPEG LLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVE VEEFVEEEDEDTV SEQ ID NO: 3 (Helix 6 Consensus Motif) Cys-Arg-Met-Val-Val-Lys-Leu-Met-Ala-Tyr-Ala-Xaa₁₂-Phe- Ala-Ser-Trp-Gly-Xaa₁₈-Xaa₁₉-Pro-Ile-Leu-Trp-Ala-Val, wherein Xaa₁₂ is Phe or Tyr, preferably wherein Xaa₁₂ is Phe; wherein Xaa₁₈ is Met or Ser, preferably wherein Xaa₁₈ is Met; and wherein Xaa₁₉ is Phe or Tyr, preferably wherein Xaa₁₉ is Phe. SEQ ID NO: 4 (Retinal binding site consensus motif) Xaa₁-Asp-Xaa₃-Xaa₄-Xaa₅-Lys-Xaa₇-Xaa₈-Xaa₉ wherein Xaa₁ is Leu, Ile, Ala, or Cys; wherein Xaa₃, Xaa₄, Xaa₅, Xaa₇, and Xaa₈ is independently any amino acid; wherein Xaa₉ is Thr, Phe, or Tyr. SEQ ID NO: 5 (Channelrhodopsin 2; ChR2; 315 aa; helix 6 highlighted in bold) MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASN VLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLY LATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGA TSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWL FFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEH ILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVNKGTGK SEQ ID NO: 6 (VChR1; accession number EU622855; 300 aa; helix 6 highlighted in bold) MDYPVARSLIVRYPTDLGNGTVCMPRGQCYCEGWLRSRGTSIEKTIAITLQWVV FALSVACLGWYAYQAWRATCGWEEVYVALIEMMKSIIEAFHEFDSPATLWLSSG NGVVWMRYGEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSA MCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVA WGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLY GDIRKKQKITIAGQEMEVETLVAEEED SEQ ID NO: 7 (ReaChR; accession number KF448069; 352 aa; helix 6 highlighted in bold) MVSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLF QTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWVVFALSVACLG WYAYQAWRATCGWEEVYVALIEMMKSIIEAFHEFDSPATLWLSSGNGVVWMRY GEWLLTCPVILIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKIL FFLISLSYGMYTYFHAAKVYIEAFHTVPKGLCRQLVRAMAWLFFVSWGMFPVLF LLGPEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKI TIAGQEMEVETLVAEEEDKYESSLE SEQ ID NO: 8 (ChR2; Helix 6 swap mutant) MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASN VLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLY LATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGA TSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRMVVKLMAYA YFASWGSYPILWAVGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHE HILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVNKGTGK SEQ ID NO: 9 (VChR1 Helix 6 swap mutant) MDYPVARSLIVRYPTDLGNGTVCMPRGQCYCEGWLRSRGTSIEKTIAITLQWVV FALSVACLGWYAYQAWRATCGWEEVYVALIEMMKSIlEAFHEFDSPATLWLSSG NGVVWMRYGEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSA MCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRMWKLMAYAYFAS WGSYPILWAVGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLY GDIRKKQKITIAGQEMEVETLVAEEED SEQ ID NO: 10 (ReaChR; Helix 6 swap mutant) MVSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLF QTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWVVFALSVACLG WYAYQAWRATCGWEEVYVALIEMMKSIIEAFHEFDSPATLWLSSGNGVVWMRY GEWLLTCPVILIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKIL FFLISLSYGMYTYFHAAKVYIEAFHTVPKGLCRMVVKLMAYAYFASWGSYPILWA VGPEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKITI AGQEMEVETLVAEEEDKYESSLE

EXAMPLE

The inventors' objective was to identify residues within the sixth transmembrane domain of Chrimson whose mutations are capable of accelerating the off-kinetics.

Example 1—Photocurrents of Chrimson Mutants in NG108-15 Cells

NG108-15 cells transiently expressing Chrimson-YFP and Chrimson-YFP mutants were investigated by patch-clamp measurements in the whole cell configuration at a clamped potential of −60 mV. The bath solution contained 140 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, pH 7.4 and the pipette solution contained 110 mM NaCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.4. Photocurrents were measured in response to 3 ms light pulses with an intensity of 23 mW/mm², and a wavelength of 594 nm. The T_(off) value was determined by a fit of the currents after cessation of illumination to a monoexponential function. The current density (J_(−60 mV)) was determined by dividing the stationary current in response to a 500 ms light pulse with an intensity of 23 mW/mm², and a wavelength of 594 nm by the capacitance of the cell.

The results are shown in FIGS. 1 and 2, and summarized in Table 1 below.

TABLE 1 Off-kinetics (T_(off)) and current density (J_(−60 mV)) of Chrimson and Chrimson mutants heterologously expressed in NG108-15 cells. Shown are the average T_(off) values (n = 3-7), the average current densities (n = 7-11) and the corresponding standard deviations. Chrimson variant τ_(off) [ms] J_(−60mV) [pA/pF] Wt 24.6 ± 0.9  24.0 ± 6.8  K176R 12.2 ± 0.8  10.1 ± 6.9  S267M 12.1 ± 1.5  22.6 ± 13.3 Y268F 11.3 ± 1.0  3.5 ± 1.6 Y261F 9.7 ± 1.5 33.3 ± 8.6  S267M/Y268F 6.3 ± 1.0 10.8 ± 5.9  Y261F/S267M 5.7 ± 0.5 34.2 ± 12.7 K176R/S267M/Y268F 4.9 ± 0.5 4.7 ± 2.7 Y261F/S267M/Y268F 3.5 ± 0.5 6.0 ± 4.7 K176R/Y261F/S267M 2.7 ± 0.3 8.3 ± 5.3 K176R/Y261F/S267M/Y268F 2.8 ± 0.3 2.6 ± 0.9

As can be taken from the above data, mutations at positions corresponding to positions 267, 268, and 261 are capable of accelerating the off-kinetics (T_(off)) of Chrimson. The combination of the mutations leads to a further acceleration of the off-kinetics (T_(off)) of Chrimson. The mutations can be advantageously combined with each other or with the known K176R mutation.

Example 2—Action Spectra of Chrimson Mutants in NG108-15 Cells

Action spectra of Chrimson and Chrimson mutants were investigated by patch-clamp measurements in the whole cell configuration at a clamped potential of −60 mV. The bath solution contained 140 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, pH 7.4 and the pipette solution contained 110 mM NaCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.4. The light-pulses with a pulse length of 7 ns were generated with the Opolette 355 tunable laser system (OPTOPRIM). For the recordings of the action spectra the pulse energies at the different wavelengths were set to values which corresponded to equal photon counts of 10¹⁸ photons/m² for Chrimson wt and 10¹⁹ photons/m² for the Chrimson mutants.

The results are shown in FIG. 3. The Y268F mutation creates a hypsochromic shift of the action spectrum (γ_(max)=580 nm). Of note, the action spectra of the tested Chrimson mutants not carrying the Y268F mutation are not significantly shifted compared to the action spectrum of wild-type Chrimson (γ_(max)=590 nm).

Example 3—Chrimson Mutants Show Increased Spiking Frequency in Rat Hippocampal Neurons

To test the Chrimson mutant's suitability for neuronal application, the construct was expressed in cultured rat hippocampal neurons.

Hippocampal Neuron Culture.

Hippocampi were isolated from postnatal P1 Sprague-Dawley rats (Jackson Laboratory) and treated with papain (20 U ml⁻¹) for 20 min at 37° C. The hippocampi were washed with DMEM (Invitrogen/Gibco, high glucose) supplemented with 10% fetal bovine serum and triturated in a small volume of this solution. ˜75,000 cells were plated on poly-D-lysine/laminin coated glass cover slips in 24-well plates. After 3 hours the plating medium was replaced by culture medium (Neurobasal A containing 2% B-27 supplement, 2 mM Glutamaxl).

Rat hippocampal neurons heterologously expressing Chrimson-YFP, Chrimson-YFP Y261F/S267M and Chrimson-YFP K176R/Y261F/S267M were investigated by patch-clamp experiments in the whole cell configuration at a clamped potential of −70 mV. Heterologous expression of wild-type Chrimson, or the respective Chrimson mutants was accomplished by transduction with adeno-associated viruses 14 to 21 days prior to the measurements.

Briefly, 1×10⁹ genome copies/ml (GC/ml) of virus was added to each well 4-9 days after plating. Expression became visible 5 days post-transduction. No neurotoxicity was observed for the lifetime of the culture (˜5 weeks). No all-trans retinal was added to the culture medium or recording medium for any of the experiments described here.

Adeno-Associated Virus (AAV2/1).

rAAV2/1 virus was prepared by the lab of Dr. Botond Roska, FMI, Basel using a pAAV2 vector with a human synapsin promoter (¹⁶) containing Chrimson-YFP wild-type or Chrimson-YFP mutants. The virus titer was nominally 1×10¹²-1×10¹³ GC/ml

Electrophysiological Recordings from Hippocampal Neurons.

For whole-cell recordings in cultured hippocampal neurons, patch pipettes with resistances of 3-8 MO were filled with 129 mM potassium gluconate, 10 mM HEPES, 10 mM KCl, 4 mM MgATP and 0.3 mM Na₃GTP, titrated to pH 7.2. Tyrode's solution was employed as the extracellular solution (125 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose and 25 mM HEPES, titrated to pH 7.4). Recordings were conducted in the presence of the excitatory synaptic transmission blockers, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX, 10 μM, Sigma) and D(−)-2-Amino-5-phosphonopentanoic acid (AP-5, 50 μM, Sigma). For determination of T_(off) and J_(−70 mV) measurements were conducted in the presence of 1 μM TTX in addition.

Electrophysiological signals were amplified using an Axopatch 200B amplifier (Axon Instruments, Union City, Calif.), filtered at 10 kHz, digitized with an Axon Digidata 1322A (50 Hz) and acquired and analyzed using pClamp9 software (Axon Instruments).

The action potentials were triggered by 40 light-pulses at indicated frequencies. The light pulses had a pulse width of 3 ms, a wavelength of λ=594 nm and a saturating intensity of 11-30 mW/mm².

The T_(off) value was determined by a fit of the currents after cessation of illumination to a monoexponential function. The current density (J_(−70 mV)) was determined by dividing the stationary current in response to a 500 ms light pulse with a saturating intensity of 20-40 mW/mm² and a wavelength of 594 nm by the capacitance of the cell. In order to determine the lowest light intensity required to induce action potentials with a probability of 100% (J₁₀₀) 40 pulses (A=594 nm, pulse width=3 ms, v=10 Hz) of varying light intensities were applied. The spike probability was calculated by dividing the number of light-triggered spikes by the total number of light pulses.

The results are shown in FIG. 4. While rat hippocampal neurons expressing wild-type Chrimson were not capable of inducing action potentials with 100% probability at frequencies above 10 Hz (cf. FIG. 4A), the Chrimson mutants Y261F/S267M (not shown) and K176R/Y261F/S267M (cf. FIG. 4B) were capable of properly inducing action potentials at 20 Hz and 40 Hz. Furthermore the Chrimson mutants K176R/Y261F/S267M (not shown) and Y261F/S267M (cf. FIG. 4C) were even able to properly induce action potentials at frequencies as high as 80-100 Hz. These measurements clearly demonstrate that the fast Chrimson mutants of the present disclosure enable rapid neural photostimulation. The measured characteristics of the Chrimson mutant K176R/Y261F/S267M, and the Chrimson mutant Y261F/S267M heterologously expressed in rat hippocampal neurons as compared to wild-type Chrimson are shown in Table 2 below.

TABLE 2 Off kinetics (T_(off)), current density (J_(−70 mV)) and the lowest light intensity required to induce action potentials with a probability of 100% (J₁₀₀). Shown are the average T_(off) (n = 3-11), the average J_(−70 mV) (n = 10-14), the average J₁₀₀ (n = 7) and the corresponding standard deviations. Chrimson J_(−70 mV) J₁₀₀ <J₁₀₀> variant τ_(off) [ms] [pA/pF] [mW mm⁻²] [mW mm⁻²] Wt 35.1 ± 9.4  40.3 ± 14.3 0.09-0.70 0.28 ± 0.19 Y261F/S267M 4.7 ± 1.8 29.9 ± 15.9 0.37-1.13 0.64 ± 0.26 K176R/Y261F/ 3.8 ± 0.4 26.5 ± 9.9  0.09-0.88 0.46 ± 0.30 S267M

As shown in Table 2 the off-kinetics (T_(off)) are significantly accelerated in the Chrimson mutant K176R/Y261F/S267M and the Chrimson mutant Y261F/S267M as compared to wild-type. Thereby the current densities (J_(−70 mV)) of Chrimson Y261F/S267M and Chrimson K176R/Y261F/S267M are only slightly reduced compared with wild-type Chrimson. The robust expression in rat hippocampal neurons enables neural photostimulation at low light intensities. These data confirm the faster kinetics as observed in NG108-15 cells (Table 1) and explain the capability of precisely inducing action potentials at high frequencies, as shown in FIG. 4 herein.

Example 4 Optogenetic Stimulation of the Auditory Pathway

Optogenetic stimulation of the auditory nerve promises a major advance in sound coding by auditory prostheses such as the cochlear implant and the auditory brainstem implant. Because light can be conveniently focused, an optical auditory prosthesis promises much improved frequency resolution of coding. One of the limitations of currently available optogenetic tools is slow kinetics relative to the speed of auditory signal processing. Here, the potential of the rapidly gating Chrimson-YFP Y261F/S267M for cochlear optogenetics was tested using postnatal AAV-mediated transduction of SGNs in mice, applying the system previously described in Hernandez et al. J Clin Invest. 2014; 124(3):1114-29. AAV2/6-hSyn-Chrimson-YFP Y261F/S267M was injected into the scala tympani via the round window in p3 mice and analyzed expression and function 4-8 weeks after injection. The injected mice did not show any overt phenotype in the colony. Chrimson-YFP Y261F/S267M expression and SGN density was analyzed using immunohistochemistry on cryosections and confocal imaging of YFP and calretinin immunofluorescence of the injected and non-injected ears (FIG. 5A, 5B). Every injected ear showed Chrimson-YFP Y261F/S267M that nearly 90% of the SGNs were transduced in the injected ear (compared to less than 5% in the non-injected ear) in all cochlear turns and SGN density was not significantly altered (FIG. 1B). The transduction rates were much higher than those achieved in our previous study with transuterine injection of AAV2/6-hSyn-CatCh-YFP (Hernandez et al., 2014) and unlike there, independent from tonotopic position. A posterior tympanotomy was performed and a 50 μm optical fiber was inserted through the round window to project the light of a 595 nm continuous wave laser on the SGN of the basal turn. We could readily elicit optical auditory brainstem responses (oABR, FIG. 6A) that were similar in amplitude and waveform to acoustic ABR (aABR). oABR amplitude grew and oABR latency got shorter with increasing light intensity (FIG. 6B, 6E). Stimuli as low as 0.5 mW (FIG. 6C, stimulus duration: 1 ms, stimulus rate: 10 s⁻¹) and as short as 80 μs (FIG. 6D, stimulus intensity: 11 mW, stimulus rate: 10 s⁻¹) were sufficient to drive oABRs. Amplitudes and waveforms of oABRs varied among the different animals (FIG. 6A) but typically varied for changes in light intensity of more than one order (FIG. 6C, output dynamic range >20 dB). oABR amplitudes decreased when decreasing stimulus duration (FIG. 6D) or raising the stimulus rates, but oABR up to 200 Hz were found (FIG. 6F). It is noted that oABR reflect a population response that extends in time over 5-7 ms and hence assume that response might have been attenuated by interaction between subsequent stimuli. It is concluded that Chrimson-YFP Y261F/S267M mediated cochlear optogenetics can drive SGNs at 175 Hz or higher, which is well in range of physiological sound-driven SGN firing rates (Liberman, M. C., J. Acoust. Soc. Am. 63, 442-455 (1978); Winter, et al., Hear. Res. 45, 191-202 (1990)). In summary, postnatal transduction of SGNs has been efficiently established, and achieved SGN firing at low light intensity and for brief stimuli. It is anticipated that Chrimson-YFP Y261F/S267M, which provides sufficient temporal fidelity and also is superior to blue channelrhodopsins for deeper light penetration and less scattering in the tissue as well as less risk of phototoxity, will become a valuable tool for optogenetic hearing restoration and auditory research.

Introducing the helix 6 motif or any other mutation disclosed herein into the constructs as described, e.g., by Hernandez et al. represents routine practice. Alternatively, one may simply replace the coding sequence of the channelrhodopsin in the conctructs by the coding sequence for the light-inducible ion channel of the present disclosure.

Example 5 Optogenetic Approach for the Recovery of Vision

Mace et al. Mol Ther. 2015; 23(1):7-16, is an earlier publication authored by some of the inventors describing optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy. Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, ON bipolar cells are targeted, which are still present at late stages of disease. For safe gene delivery, a recently engineered AAV variant is used that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. It is shown that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice.

Introducing the helix 6 motif identified herein into the constructs as described, e.g., by Macé et al. represents routine practice. Alternatively, one may simply replace the coding sequence of the channelrhodopsins in the constructs by the coding sequence for the light-inducible ion channel of the present disclosure. The new light-inducible ion channels of the present disclosure are inserted in the cassettes for the activation of ON bipolar cells as well as for the Ganglion cells in the retina.

LIST OF REFERENCES

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The invention claimed is:
 1. A mutant light-inducible ion channel, wherein the mutant light-inducible ion channel comprises an amino acid sequence selected from the group consisting of A and B: A: an amino acid sequence which has at least 95% identity to the full length sequence of SEQ ID NO: 1 (Chrimson), wherein the mutant light-inducible ion channel only differs from its parent light-inducible ion channel by a substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1, which substitution(s) accelerate(s) the off-kinetics of the mutant channel as compared to the parent channel, when compared by patch-clamp measurements in the whole cell configuration at a clamp potential of −60 mV, a bath solution of 140 mM NaCl, 2 mM CaCl₂, 2 MgCl₂, 10 mM HEPES, pH 7.4, and a pipette solution of 110 mM NaCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.4; and B: an amino acid sequence identical to the sequence of SEQ ID NO: 2 (CsChrimson), except for a substitution at one or more amino acid position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1, and optionally an Arg at the position corresponding to K176 of SEQ ID NO: 1; and wherein said substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1 is one or more of the following: a Phe at the amino acid position corresponding to position Y261 of SEQ ID NO: 1; a Phe at the amino acid position corresponding to position Y268 of SEQ ID NO: 1; a Met at the amino acid position corresponding to position S267 of SEQ ID NO:
 1. 2. The mutant light-inducible ion channel of claim 1, wherein the amino acid sequence comprised by the mutant light-inducible ion channel has at least 96%, at least 97%, at least 98%, or at least 99% identity to the full length of SEQ ID NO: 1 (Chrimson).
 3. The mutant light-inducible ion channel of claim 1, wherein the mutant light-inducible ion channel comprises a Phe at the amino acid positions corresponding to position Y261 and a Met at the amino acid position corresponding to position S267 of SEQ ID NO: 1; or wherein the mutant light-inducible ion channel comprises a Phe at the amino acid positions corresponding to position Y268 and a Met at the amino acid position corresponding to position S267 of SEQ ID NO:
 1. 4. The mutant light-inducible ion channel of claim 1, wherein the mutant light-inducible ion channel comprises a Phe at the amino acid positions corresponding to position Y261, a Phe at the amino acid position corresponding to position Y268, and a Met at the amino acid position corresponding to position S267 of SEQ ID NO:
 1. 5. The mutant light-inducible ion channel of claim 1, further comprising an Arg at the position corresponding to position 176 of SEQ ID NO:
 1. 6. The mutant light-inducible ion channel of claim 1, wherein the mutant channel comprises the motif of SEQ ID NO: 3: Cys-Arg-Met-Val-Val-Lys-Leu-Met-Ala-Tyr-Ala-Xaa₁₂-Phe-Ala-Ser-Trp-Gly-Xaa₁₈-Xaa₁₉-Pro-Ile-Leu-Trp-Ala-Val, wherein Xaa₁₂ is Phe; wherein Xaa₁₈ is Met; and wherein Xaa₁₉ is Phe or Tyr.
 7. The mutant light-inducible ion channel of claim 1, wherein the mutant light-inducible ion channel comprises the amino acid sequence of SEQ ID NO: 1 (Chrimson), except for said substitution(s) at position Y261, Y268, and S267, and optionally the Arg at the position corresponding to position 176 of SEQ ID NO:
 1. 8. The mutant light-inducible ion channel of claim 1, wherein the mutant light-inducible ion channel is a red light absorbing channelrhodopsin.
 9. A nucleic acid construct, comprising a nucleotide sequence coding for a mutant light-inducible ion channel, wherein the mutant light-inducible ion channel comprises an amino acid sequence selected from the group consisting of A and, B and a combination thereof: A: an amino acid sequence which has at least 95% identity to the full length sequence of SEQ ID NO: 1 (Chrimson), wherein the mutant light-inducible ion channel only differs from its parent light-inducible ion channel by a substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1, which substitution(s) accelerate(s) the off-kinetics of the mutant channel as compared to the parent channel, when compared by patch-clamp measurements in the whole cell configuration at a clamp potential of −60 mV, a bath solution of 140 mM NaCl, 2 mM CaCl₂), 2 MgCl₂, 10 mM HEPES, pH 7.4, and a pipette solution of 110 mM NaCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.4; and B: an amino acid sequence identical to the sequence of SEQ ID NO: 2 (CsChrimson), except for a substitution at one or more amino acid position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1, and optionally an Arg at the position corresponding to K176 of SEQ ID NO: 1; and wherein said substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1 is one or more of the following: a Phe at the amino acid position corresponding to position Y261 of SEQ ID NO: 1; a Phe at the amino acid position corresponding to position Y268 of SEQ ID NO: 1; a Met at the amino acid position corresponding to position S267 of SEQ ID NO:
 1. 10. An expression vector, comprising a nucleotide sequence coding for a light-inducible ion channel, wherein the mutant light-inducible ion channel comprises an amino acid sequence selected from the group consisting of A and, B and a combination thereof: A: an amino acid sequence which has at least 95% identity to the full length sequence of SEQ ID NO: 1 (Chrimson), wherein the mutant light-inducible ion channel only differs from its parent light-inducible ion channel by a substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1, which substitution(s) accelerate(s) the off-kinetics of the mutant channel as compared to the parent channel, when compared by patch-clamp measurements in the whole cell configuration at a clamp potential of −60 mV, a bath solution of 140 mM NaCl, 2 mM CaCl₂), 2 MgCl₂, 10 mM HEPES, pH 7.4, and a pipette solution of 110 mM NaCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.4; and B: an amino acid sequence identical to the sequence of SEQ ID NO: 2 (CsChrimson), except for a substitution at one or more amino acid position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1, and optionally an Arg at the position corresponding to K176 of SEQ ID NO: 1; and wherein said substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1 is one or more of the following: a Phe at the amino acid position corresponding to position Y261 of SEQ ID NO: 1; a Phe at the amino acid position corresponding to position Y268 of SEQ ID NO: 1; a Met at the amino acid position corresponding to position S267 of SEQ ID NO:
 1. 11. A cell comprising the nucleic acid construct according to claim
 10. 12. A high-throughput screening method comprising the following steps: (i) providing a cell expressing a Ca++-inducible potassium channel and the light-inducible ion channel of claim 1, (ii) applying a light stimulus in order to induce the light-inducible ion channel, (iii) determining the alteration of the membrane potential, and (iv) comparing the signal determined in step (iii) with the signal determined in a cell in the presence of a candidate agent directed against the Ca++-inducible potassium channel subjected to step (ii), wherein a difference in the change of the membrane potential is indicative of the candidate agent being a modulator of the Ca++-inducible potassium channel.
 13. A method for light-stimulation of neuron cells comprising applying a light stimulus to a cell comprising a light-inducible ion channel according to claim
 1. 14. A non-human animal, comprising a light-inducible ion channel according to claim
 1. 15. The cell according to claim 11, wherein the cell is a mammalian cell.
 16. The cell according to claim 15, wherein the cell is selected from the group consisting of (a) a hippocampal cell, a photoreceptor cell, a retinal rod cell, a retinal cone cell, a retinal ganglion cell, a bipolar neuron, a ganglion cell, a pseudounipolar neuron, a multipolar neuron, a pyramidal neuron, a Purkinje cell, or a granule cell; and (b) a neuroblastoma cell, a HEK293 cell; a COS cell; a BHK cell; a CHO cell; a myeloma cell; or a MDCK cell.
 17. The cell according to claim 16, wherein the cell is a NG108-15 neuroblastoma cell. 